The one year survival on gemcitabine, the drug of choice for pancreatic cancer, is about 20% and the overall survival of this patient population is only 4%. The molecular mechanisms underlying the incomplete and differential response to this chemotherapy is currently not known, but are suspected to involve genetic aberrations that accumulate during progression. It is known that common regions of the genome undergo high-level DNA amplification in pancreatic cancer. These regions likely harbor oncogenes whose over-expression provide the cancer cell with growth and survival advantages. The central hypothesis for the proposed research is that specific gene expression changes, driven by DNA amplification, modulate the sensitivity of pancreatic tumor cells to gemcitabine. We expect that a parallel RNAi approach can be applied to greatly accelerate the experimental validation of this hypothesis in multiple amplified candidate genes. Specific aims: (1) Prioritize Amplified and Over-Expressed Genes in Pancreatic Cancer which are associated with gemcitabine sensitivity. (2) Validate the functional relevance by determining the extent to which experimental manipulation of specific gene expression can modulate sensitivity to gemcitabine. (3) Validate the clinical relevance by high throughput analysis of gene copy number and protein expression of sensitizing targets on a pancreatic cancer tissue microarray. This research is relevant and significant to human because it addresses an urgent clinical problem related to incomplete and variable response to chemotherapy in pancreatic cancer. The goal of this innovative research proposal will be to validate the functional role and clinical relevance of amplified genes in modulating sensitivity to gemcitabine. Using advanced technologies, we expect to generate this valuable outcome with unprecedented speed. The products of this project will be advanced through the other drug development components of this program to directly generate and advance new therapeutics.